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2 www.epicentre.com
In Vivo Transposomics™ Research
EZ-Tn5™ <KAN-2>Tnp and EZ-Tn5™ <DHFR-1>Tnp Transposome™ KitsRapidly generate knock-in or knock-out mutations
EZ-Tn5 Transposome complexes are formed between an EZ-Tn5 Transposon and EZ-Tn5 Transposase, and provide a simple and reliable method for generating a library of random gene knockouts in vivo.* The kits can also be used for knock-in of genes for bacterial strain development, tagging bacteria for environmental localization studies, and direct sequencing of bacterial chromosomal DNA.
Just electroporate the EZ-Tn5 Transposome into any of a broad range of living bacterial cells and select for a marker encoded by the EZ-Tn5 Transposon (Fig. 1).
• Rapid mutagenesis procedure is simpler and easier to use than chemical mutagenesis.• More efficient than using mini-transposons with suicide plasmids.• Broad host range: over 60 species of Gram-negative and Gram-positive bacteria
transposed so far.
*The use of Transposome™ complexes for in vivo insertion of a transposon, including, but not limited, to HyperMu™ and EZ-Tn5™ Transposome™ complexes, is covered by U.S. Patent No. 6,159,736 and related patents and patent applications, exclusively licensed to Epicentre. See www.epicentre.com/legal.
EZ-Tn5™Transposome
Cells
Sequencing
Insertion Clones1. Electroporate
2. PlateSelection Plate
Genomic DNA
Figure1.Overviewofin vivotransposition.
The EZ-Tn5™ Transposon insertion site in bacterial DNA can be sequenced directly using genomic DNA isolated using the MasterPure™ Complete DNA Purification Kit and primers homologous to the ends of the transposon.
Cat.# Quantity
EZ-Tn5™ <KAN-2>Tnp Transposome™ KitTSM99K2 10 Reactions
EZ-Tn5™ <DHFR-1>Tnp Transposome™ KitTSM99D1 10 ReactionsContents: Ready-to-use Transposome complex with either a KAN-2 or DHFR-1 marker, and forward and reverse primers for sequencing.
[email protected] • (800) 284-8474 3
In Vivo Transposomics™ Research
Cat.# Quantity
EZ-Tn5™ <R6Kγori/KAN-2>Tnp Transposome™ KitTSM08KR 10 ReactionsContents: EZ-Tn5 <R6Kγori/KAN-2>Tnp Transposome, KAN-2 FP-1 Forward Primer, R6KAN-2 RP-1 Reverse Primer, Sterile Water.
Figure1.Overviewofrescuecloningoftransposoninsertionsites.
<R6Kγori/KAN-2> clone Purify, then shear or digest genomic DNA
Self-ligation
RKan
ori
Rescued plasmid DNA KanR rescued clones
Transform pir E. coli and select on Kan plates
RKanori
RKan
ori
RKanori
RKanori
RKanori
RKanori
EZ-Tn5™ <R6Kγori/KAN-2>Tnp Transposome™ KitSimplify rescue cloning of genomic DNA or plasmids
Nothing makes the cloning process easier than creating mutations in vivo with the EZ-Tn5 <R6Kγori/KAN-2>Tnp Transposome Kit.* In addition to encoding a broad host-range kanamycin-resistance gene, the transposon contains an E. coli conditional origin of replication (R6Kγori). The presence of this origin of replication enables the propagation or “rescue” of the region of genomic DNA, or plasmid, into which the transposon has been inserted (Fig. 1).
• Easily recover and propagate plasmids from diverse bacterial genera that will not normally replicate in E. coli.
• Simple rescue cloning process of mutagenized genes speeds up structure/function studies and sequencing.
• Random insertion of transposon DNA assures excellent coverage of the entire bacterial chromosome.
• Rescue clones can be sequenced bidirectionally using the primers provided that are homologous to the ends of the transposon.
*The use of Transposome™ complexes for in vivo insertion of a transposon, including, but not limited, to HyperMu™ and EZ-Tn5™ Transposome™ complexes, is covered by U.S. Patent No. 6,159,736 and related patents and patent applications, exclusively licensed to Epicentre. See www.epicentre.com/legal.
4 www.epicentre.com
In Vivo Transposomics™ Research
EZ-Tn5™ Custom Transposome™ Construction KitsCreate your own EZ-Tn5 Transposomes using almost any DNA
The new EZ-Tn5 Custom Transposome Construction Kits provide the key reagents needed for constructing your own Transposomes. Also included are the standard pMOD PCR primers, 10X EZ-Tn5 Reaction Buffer for in vitro reactions, detailed protocols for creating and purifying the custom transposons, and stop solution for in vitro reactions. Sufficient reagents are provided to create Transposomes for up to 10 in vitro or 20 in vivo transposition reactions.
Cat.# Quantity
EZ-Tn5™ Custom Transposome Construction Kit with pMOD-2 VectorTNP10622 20 reactions
EZ-Tn5™ Custom Transposome Construction Kit with pMOD-3 VectorTNP10623 20 reactions
TypeOne™ Restriction InhibitorIncrease transformation efficiency of most bacterial strains
DNA transformation can be difficult to achieve in many bacterial strains due to the presence of one or more restriction and modification (R-M) systems that cleave unmodified DNA that is recognized as “foreign.” TypeOne™ Restriction Inhibitor* provides a powerful method for increasing transformation efficiencies in bacterial strains with type I R-M systems. Type One Restriction Inhibitor also increases the insertion efficiency of EZ-Tn5 Transposome complexes. It has also been reported to block Type III restriction systems.
• Easy to use: Simply add TypeOne Restriction Inhibitor to unmodified DNA or a Transposome and electroporate using standard methods.
• Blocks all known families of type I R-M enzymes and can therefore be used to increase transformation efficiencies in a variety of bacterial cells.
*Covered by issued and/or pending patents. See www.epicentre.com/legal.
Cat.# Quantity
TypeOne™ Restriction InhibitorTY0261H 100 µg
[email protected] • (800) 284-8474 5
In Vivo Transposomics™ Research
Figure1.OverviewoftheprocedurefortheEZ-Tn5™CustomTransposome™ConstructionKit.
Target DNA
Transposon Transposase
In Vitro Use
In Vivo Use
EZ-Tn5™Transposon
EZ-Tn5™Transposome™
EZ-Tn5™Transposome™
Cells
1. Electroporate2. Plate
Insertion Clones
Phenotypic Analysis
Direct GenomicDNA Sequencing
Rescue Cloning
10 min, 37°C
Selection Plate
1. Incubate 37°C; 2 hrs2. Transform E. coli3. Select Drugr clones
pMOD™-3<R6Kγori/MCS>
ForwardPCR Primer
ForwardSequencing
PrimerReverse
PCR Primer
ME MEPvu IIPshAI
Pvu IIPshAI
pUC-19 MultipleCloning SiteEcoRI HindIII
R6Kγ ori
ReverseSequencing
Primer
Vector( )colE1 ori, AmpR
pMOD™-2<MCS>
ForwardPCR Primer
ForwardSequencing
PrimerReverse
PCR Primer
ME MEPvu IIPshAI
Pvu IIPshAI
pUC-19 MultipleCloning SiteEcoRI HindIII
ReverseSequencing
Primer
Vector( )colE1 ori, AmpR
4. Prepare template DNA
minus Mg++
MEME
6 www.epicentre.com
In Vitro Transposon Insertion Kits
EZ-Tn5™ <KAN-2>, EZ-Tn5™ <TET-1>, and EZ-Tn5™ <DHFR-1> Insertion KitsCompletely sequence clones without subcloning or primer walking
EZ-Tn5 Insertion Kits* are designed to simplify and speed up complete sequencing of any cloned DNA >2 kb, without primer walking or subcloning. A simple, one-step in vitro reaction results in random insertion of a single EZ-Tn5 Transposon containing a selectable marker into the clone. Then, transform E. coli cells with an aliquot of the reaction and select for the marker encoded by the EZ-Tn5 Transposon. Use the primers provided in the kits to sequence insertion clones bidirectionally from primer binding sites at the ends of the inserted transposon.
• Primer binding sites are distributed throughout the clone, ensuring much better sequence coverage compared to primer walking or subcloning.
• A single reaction generates up to 106 insertion clones—enough to sequence even the largest clone. Save time usually spent subcloning or designing and synthesizing sequencing primers.
• Use a single set of sequencing primers (provided in the kits) to completely sequence any clone.
• The EZ-Tn5 System can be used for processing multiple DNA samples, making it an effective component of a high-throughput sequencing pipeline.
*Covered by intellectual property rights licensed to Epicentre.
Cat.# Quantity
EZ-Tn5™ <KAN-2> Insertion Kit EZI982K 10 Reactions
EZ-Tn5™ <TET-1> Insertion Kit EZI921T 10 Reactions
EZ-Tn5™ <DHFR-1> Insertion Kit EZI912D 10 Reactions
Contents: EZ-Tn5 Transposase, EZ-Tn5 Transposon, EZ-Tn5 10X Reaction Buffer, EZ-Tn5 10X Stop Solution, Forward and Reverse Sequencing Primers, Control Target DNA, Sterile Water.
[email protected] • (800) 284-8474 7
In Vitro Transposon Insertion Kits
EZ-Tn5™ <oriV/KAN-2> Insertion Kit The EZ-Tn5 <oriV/KAN-2> Insertion Kit* randomly inserts a single EZ-Tn5™ <oriV/KAN-2> Transposon containing an inducible, high-copy origin of replication (oriV) into the target DNA. Thus, the kit integrates CopyControl™ capability into those low- to single-copy-number clones (such as BACs or fosmids) that do not possess this feature. Selected clones are amplified by adding the CopyControl Induction or Autoinduction Solution, and microgram quantities of template DNA can be purified from as little as 1 ml of culture.
EZ-Tn5™ <T7/KAN-2> Promoter Insertion KitThe EZ-Tn5 <T7/KAN-2> Promoter Insertion Kit* provides an easy and reliable method to randomly insert a transposon containing a phage T7 RNA polymerase promoter into any DNA molecule in vitro.
EZ-Tn5™ <R6Kγori/KAN-2> Insertion KitThe EZ-Tn5 <R6Kγori/KAN-2> Insertion Kit* facilitates the sequencing and genetic analysis of plasmids or any other circularized DNA that would not otherwise replicate in E. coli. The kit is based upon the EZ-Tn5 <R6Kγori/KAN-2> Transposon, which carries the E. coli R6Kγ conditional origin of replication (R6Kγori) and a kanamycin- resistance marker. Clones can be maintained in Epicentre’s TransforMax™ EC100D™ pir+ or TransforMax™ EC100D™ pir-116 strains.*Covered by intellectual property rights licensed to Epicentre.
Cat.# Quantity
EZ-Tn5™ <oriV/KAN-2> Insertion KitEZI02VK 10 ReactionsContents: EZ-Tn5 Transposase, EZ-Tn5 <oriV/KAN-2> Transposon, EZ-Tn5 10X Reaction Buffer, EZ-Tn5 10X Stop Solution, Forward and Reverse Primers, Control Target DNA, Sterile Water.
EZ-Tn5™ <T7/KAN-2> Promoter Insertion KitEZI03T7 10 ReactionsContents: EZ-Tn5 Transposase, EZ-Tn5 <T7/KAN-2> Transposon, EZ-Tn5 10X Reaction Buffer, EZ-Tn5 10X Stop Solution, Forward and Reverse Primers, Control Target DNA, Sterile Water.
EZ-Tn5™ <R6Kγori/KAN-2> Insertion KitEZI011RK 10 ReactionsContents: EZ-Tn5 Transposase, EZ-Tn5 <R6Kγori/KAN-2> Transposon, EZ-Tn5 10X Reaction Buffer, EZ-Tn5 10X Stop Solution, Forward and Reverse Primers, Control Target DNA, Sterile Water.
8 www.epicentre.com
DNA Purification
Cat.# Quantity
Meta-G-Nome™ DNA Isolation Kit MGN0910 10 PurificationsContents: Extraction buffer, Filter Wash Buffer, Meta-Lysis Solution (2X), MPC Protein Precipitation Reagent, Proteinase K, Fosmid Control DNA, RNase A, Ready-Lyse Lysozyme Solution, TE Buffer, 0.45-µm cellulose acetate filter membranes, 1.2-µm cellulose acetate filter membranes.
Meta-G-Nome™ DNA Isolation KitPurify metagenomic DNA from water or soil samples
The Meta-G-Nome DNA Isolation Kit is used to isolate DNA from unculturable or difficult-to-culture microbial species present in environmental water or soil samples. The simple extraction procedure involves low-speed centrifugation prior to filtration, and lysis with Ready-Lyse™ Lysozyme and Proteinase K. The kit isolates high-molecular-weight DNA that is randomly sheared (with fragment sizes approximately 40 kb), does not require size selection, and can be used directly for end-repair and construction of fosmid libraries. The kit can also be used to prepare 16S rRNA metagenomic libraries for phylogenetic classification or species-abundance studies.
• Isolated DNA is also suitable for PCR (Fig. 1) or sequencing.• No bead-beating, agarose plugs, pulse-field gel electrophoresis, or size selection.• No phenol/chloroform extractions or CTAB.
M 1 2 3 4 5 6DNA was isolated following the kit protocol, and PCR was performed using 16S rRNA primers using various dilutions of the template DNA. Lane M, Kilobase ladder; lanes 1 and 2, 1 µl and 5 µl of PCR products from 1:10 template dilution; lanes 3 and 4, 1 µl and 5 µl of PCR products from 1:100 template dilution; lane 5, 1 µl of PCR products from undiluted template; lane 6, 1:10 dilution of sample from lane 5.
Figure1.PCRamplificationofsoilmetagenomicDNA.
[email protected] • (800) 284-8474 9
DNA Purification
MasterPure™ Complete DNA and RNA Purification KitPurify TNA, DNA, or RNA from any source
The MasterPure Complete DNA and RNA Purification Kit enables efficient purification of intact total nucleic acid (TNA), DNA, or RNA from every type of biological material. The purified DNA is suitable for sequencing and other molecular biology applications.
• Purify DNA in 30 minutes without spin columns.• A260/A280 ratios consistently between 1.8 and 2.0.• Recover >90% of theoretical DNA yield, including both low- and high-molecular-
weight nucleic acids.• No phenol, chloroform, or other caustic solvents.
Cat.# Quantity
MasterPure™ Complete DNA and RNA Purification KitMC89010 5/10 PurificationsMC85200 200 PurificationsFor isolating TNA, DNA, or RNA.Contents: Red Cell Lysis Solution, Tissue and Cell Lysis Solution, MPC Protein Precipitation Reagent, 2X T&C Lysis Solution, TE Buffer, RNase A, RNase-Free DNase I, Proteinase K, 1X DNase Buffer, RiboGuard RNase Inhibitor.Note: Cat. # MC89010 contains reagents for 10 TNA, 10 DNA, or 5 RNA purifications and Cat. # MC85200 contains reagents for 200 TNA, 200 DNA, or 100 RNA purifications.
Sample SampleSize TNAµg DNAµg RNAµgHeLa/HL60 cells 1 x 106 cells 10-30 3-12 7-15TissuesLiver 5 mg 33-42 5-10 13-25Brain 5 mg 9-13 6-9 4-11Heart 5 mg 6-10 4-7 4-5Kidney 5 mg 10-17 3-8 14-17Thymus 5 mg 15-30 6-12 9-18Blood 200 µl 3-10 3-9Buffy coat 300 µl 40-55 40-55 3-6Mouse tail 0.5 cm 25-30 9-11 E. coli 3.5 x 106 cells 2.5-2.8 1.3-1.6 1.6-1.8S. mutans 1.5 ml 0.9 Yeast* 2.2 x 106 cells 11-18(S. cerevisiae) 1.1 x 107 cells 70-78
*For extraction of yeast DNA, the MasterPure™ Yeast DNA Purification Kit is recommended.
Table1.TypicalyieldsobtainedusingtheMasterPure™CompleteKit.
10 www.epicentre.com
DNA Purification
MasterPure™ Gram-Positive DNA Purification KitPurify genomic DNA from Gram-positive bacteria
The MasterPure Gram-Positive DNA Purification Kit provides all of the reagents needed to purify DNA from Gram-positive bacteria. The purified DNA is suitable for fosmid library construction, PCR, restriction digests, Southern blotting, and other molecular biology applications. The protocol can also be adapted for total RNA purification.
• Scalable method for large sample volumes.• Yields high-quality, high-molecular-weight DNA.• Lysozyme included; no separate purchase required.• Tested on a variety of species.• Can also be used with Gram-negative bacteria.
Cat.# Quantity
MasterPure™ Gram-Positive DNA Purification KitMGP04020 20 ReactionsMGP04100 100 Reactions
Contents: Gram-Positive Cell Lysis Solution, MPC Protein Precipitation Reagent, Ready-Lyse
Lysozyme, Proteinase K, TE Buffer, RNase A.
DNA purified from B. subtilis (ATCC 6051) was separated on a 1% agarose gel and stained with SYBR® Gold. Lane M, kilobase ladder; lane 1, 300 ng of B. subtilis DNA.
12 kb
1 kb
M 1
Figure1.ElectrophoreticanalysisofDNApurifiedusingtheMasterPure™Gram-PositiveDNAPurificationKit.
[email protected] • (800) 284-8474 11
DNA Purification
MasterPure™ Yeast DNA Purification KitPurify DNA at high yields from yeast
The MasterPure Yeast DNA Purification Kit enables efficient, high-yield purification of high-molecular-weight DNA from yeast and other fungi. The protocol involves nonenzymatic cell lysis at 65°C, followed by removal of protein by precipitation, and nucleic acid precipitation and resuspension. The protocol can be easily scaled for larger or smaller samples, including single yeast colonies. The recovered nucleic acid can be used directly in most applications, including sequencing, PCR amplification, restriction endonuclease digestion, Southern blotting, and genomic library preparation.
• Higher yields of yeast chromosomal DNA than with other kits (Fig. 1).• Recover DNA from a wide variety of yeast species, including Candida, Saccharomyces,
Pichia, and Schizosaccharomyces, and filamentous fungi such as Aspergillus and Penicillium.
• Purify high-molecular-weight DNA in less than 40 minutes.• No bead-beating, columns, or phenol or other organic extractions.
Cat.# Quantity
MasterPure™ Yeast DNA Purification KitMPY80010 10 PurificationsMPY80200 200 PurificationsContents: Yeast Cell Lysis Solution, MPC Protein Precipitation Reagent, TE Buffer, RNase A.
DNA was quantitated specifically with Hoechst fluorescent dye 33258, which gives minimal fluorescence with RNA. The data represent the average DNA yields determined by fluorometry from two experiments with S. cerevisiae and C. albicans. The MasterPure Kit produced up to 17 times more DNA from C. albicans and 12 times more DNA from S. cerevisiae than other kits.
0 1,000 2,000 3,000 4,000 5,000 6,000 7,000 8,000DNA, nanograms
MasterPure™ Kit – Supplier Q – Supplier F – MasterPure™ Kit –
Supplier Q – Supplier F –
C. albicans
S. cerevisiae
Figure1.ComparativeyieldsofDNA.
12 www.epicentre.com
RNA Purification
MasterPure™ Yeast RNA Purification KitPurify RNA from yeast and filamentous fungi
The MasterPure Yeast RNA Purification Kit provides all of the reagents needed to purify RNA from yeast cell types (including Candida, Saccharomyces, Schizosaccharomyces, Pichia) and filamentous fungi. The kit uses a rapid salting-out process to remove contaminating macromolecules. The purified RNA is suitable for cDNA synthesis and microarray gene expression analysis.
• No hot acid phenol, spin columns, or bead-beating.• Faster than spheroplasting.• No extra enzymes or equipment to purchase.• Yields 25-50 µg RNA from 1 ml of mid-log S. cerevisiae.
Cat.# Quantity
MasterPure™ Yeast RNA Purification KitMPY03010 10 ReactionsMPY03100 100 ReactionsContents: Extraction Reagent for RNA, MPC Protein Precipitation Reagent, TE Buffer (in 100-reaction kit only), Proteinase K, RNase-Free DNase I, RiboGuard RNase Inhibitor, 10X DNase Buffer, 2X T & C Lysis Solution.
RNA was extracted from S. cerevisiae using the MasterPure™ Kit and stored at –20°C for 2 months before analysis on an Agilent Technologies 2100 Bioanalyzer. The electropherogram demonstrates the high-purity, intact RNA.
Figure1.PurityofRNA.
[email protected] • (800) 284-8474 13
DNA Extraction
QuickExtract™ Bacterial DNA Extraction KitExtract DNA from Gram-positive and Gram-negative Bacteria
The QuickExtract Bacterial DNA Extraction Kit provides a simple method for extracting DNA from Gram-positive and Gram-negative bacteria. The kit contains QuickExtract Bacterial DNA Extraction Solution and Ready-Lyse™ Lysozyme Solution, a proven reagent with over 200 times the specific activity of egg-white lysozyme. After mixing and incubating for 15 minutes, the isolated DNA is ready for PCR or other downstream applications such as restriction digests, PFGE (pulsed-field gel electrophoresis), and optical mapping. The kit has been tested on a range of bacteria (Table 1) and has been shown to produce very long DNA strands (Fig. 1).
• Fast—PCR-ready DNA in 15 minutes, ideal for high-throughput workflows.• Safe—No organic extraction.• Efficient—No columns, transfers, or sample loss.
Cat.# Quantity
QuickExtract™ Bacterial DNA Extraction KitQEB0905T 5 ml (50 Extractions)QEB09050 50 ml (500 Extractions)
Contents: QuickExtract Bacterial DNA Extraction Solution, Ready-Lyse Lysozyme Solution.
Gram-positive Gram-negativeBacillus subtilis E. coliBifidobacterium spp Salmonella entericaBrevibacterium linens Salmonella typhimuriumClostridium perfringens Vibrio gazogenesLactobacillus plantarum Listeria monocytogenes Staphylococcus equorum Streptococcus agalactiae Streptococcus pyogenes Streptococcus thermophilus
Table1.SpeciesofbacteriathathavebeentestedwiththeQuickExtract™BacterialDNAExtractionKit.
Many strands are 400-500 kb. Image courtesy of Dr. Shiguo Zhou, University of Wisconsin-Madison.
~250 kb
Figure1.LongDNAstrandsfromE. coliextractedusingtheQuickExtract™BacterialDNAExtractionKit.
14 www.epicentre.com
DNA-Seq
End-It™ DNA End-Repair KitRepair DNA ends for adaptor ligation
The End-It DNA End-Repair Kit converts DNA containing damaged or incompatible 5′- and/or 3′-protruding ends to 5′-phosphorylated, blunt-ended DNA. The end-repaired DNA can be used for ligation into a cloning vector or ligation of next-generation sequencing adaptors, using the Fast-Link™ DNA Ligation Kit. The high specific activity of the End-Repair Enzyme Mix provides complete conversion of protruding ends to 5′-phosphorylated, blunt-ended DNA.
The End-It DNA End-Repair Kit contains reagents for 20 or 50 end-repair reactions (repair of up to 100 µg or 250 µg of genomic DNA, respectively).
Cat.# Quantity
End-It™ DNA End-Repair KitER0720 20 ReactionsER81050 50 ReactionsContents: End-Repair Enzyme Mix, End-Repair 10X Buffer, dNTP Solution, ATP.
Fast-Link™ DNA Ligation KitLigate next-generation sequencing adaptors to DNA
The Fast-Link DNA Ligation Kit uses a high-quality ligase (Fast-Link T4 DNA Ligase), to provide extremely rapid, high-efficiency DNA ligation. Cohesive-end ligations can be performed in 5 minutes at room temperature. In contrast to other ligases, it is not necessary to desalt Fast-Link ligation reactions prior to transformation of electrocompetent or chemically competent cells. The Fast-Link Kit can be used for routine and high-throughput DNA cloning.
Cat.# Quantity
Fast-Link™ DNA Ligation KitLK11025 25 LigationsLK0750H 50 LigationsLK6201H 100 LigationsContents: Fast-Link DNA Ligase, Fast-Link 10X Ligation Buffer, 10 mM ATP.
[email protected] • (800) 284-8474 15
DNA-Seq
CircLigase™ and CircLigase II ssDNA LigasesCircularize ssDNA templates
CircLigase ssDNA Ligase* is a thermostable ATP-dependent ligase that catalyzes intramolecular ligation (i.e., circularization) of ssDNA templates having a 5′-phosphate and a 3′-hydroxyl group. In contrast to other ligases, CircLigase ssDNA Ligase ligates ends of ssDNA in the absence of a complementary sequence. The enzyme is therefore useful for making circular ssDNA molecules from linear ssDNA. Circular ssDNA molecules can be used as substrates for rolling-circle replication or rolling-circle transcription.
CircLigase II ssDNA Ligase* has greater activity and a new Reaction Buffer for improved ligation efficiency.
Unit Definition: One unit of CircLigase enzyme converts 1 pmol of a linear 5′-phosphorylated CircLigase Standard 55-mer Oligo into an Exonuclease I–resistant circular form in 1 hour at 60°C under standard assay conditions.*Covered by intellectual property rights licensed to Epicentre.
Cat.# Quantity
CircLigase™ ssDNA LigaseCL4111K 1,000 Units CL4115K 5,000 UnitsContents: CircLigase ssDNA Ligase, CircLigase 10X Reaction Buffer, 1 mM ATP, 50 mM MnCl2, CircLigase ssDNA Control Oligo, Sterile Water.
CircLigase™ II ssDNA LigaseCL9021K 1,000 Units CL9025K 5,000 UnitsContents: CircLigase II ssDNA Ligase, CircLigase II 10X Reaction Buffer, 50 mM MnCl2, CircLigase ssDNA Control Oligo, Betaine, Sterile Water.
DisplaceAce™ DNA Polymerase Improve DNA sequencing of GC-rich regions
DisplaceAce DNA Polymerase* is a recombinant DNA polymerase modified to remove 5′→3′ exonuclease activity. It has strong strand-displacing DNA polymerase activity, similar to that of Bacillus DNA polymerases. The DNA-dependent DNA polymerase activity is optimal at approximately 65°C. It also has RNA-dependent DNA polymerase activity. The enzyme can be inactivated by incubation at 80°C for 20 minutes.
Unit Definition: One unit converts 10 nmol of dNTPs into acid-insoluble material in 30 minutes at 65°C under standard assay conditions.*Covered by issued and/or pending patents.
Cat.# Concentration Quantity
DisplaceAce™ DNA PolymeraseD090710K 100U/ul 10,000 Units
16 www.epicentre.com
rRNA Removal
RiboZero™ rRNA Removal KitsRemove up to 99.9% of the rRNA from a total RNA sample
The Ribo-Zero™ rRNA Removal Kits remove more ribosomal RNA (rRNA) from total RNA preparations than any other method. The kits are ideal for preparing RNA samples for whole-transcriptome RNA-Seq, random-primed cDNA synthesis, and other RNA analysis applications.
• Suitable for both intact and partially degraded RNA.• Recovers mRNA and small and large ncRNAs for whole-transcriptome RNA-Seq.• Single-pass, 90- to 120-minute process.• Recovers all fragments of nonribosomal RNAs in a partially degraded RNA sample.
Cat.# Quantity
Ribo-Zero™ rRNA Removal Kit (Meta-Bacteria)RZMB11086 6 Reactions
Ribo-Zero™ rRNA Removal Kit (Gram-Negative Bacteria)RZNB1056 6 Reactions
Ribo-Zero™ rRNA Removal Kit (Gram-Positive Bacteria)RZPB10106 6 Reactions
InputRNA
PercentRemovalofrRNA
23S 16S 5S
Partially degraded B. subtilis total RNA
>99.9% >99.9% 93.5%
Intact E. coli total RNA >99.9% >99.9% >97%
Table1.RNAremovalwithRibo-Zero™KitsdeterminedbyqPCR.
[email protected] • (800) 284-8474 17
RNA-Seq
Cat.# Quantity
ScriptSeq™ mRNA-Seq Library Preparation Kit (Illumina®-Compatible)SS10906 6 ReactionsSS10924 24 Reactions
RNA-Seq Barcode Primers (Illumina®-Compatible)RSBC10948 48 Reactions eachSet of 12 Illumina®-compatible barcodes.
ScriptSeq™ mRNA-Seq Library Preparation KitsDirectional mRNA-Seq libraries in 3 hours
The ScriptSeq mRNA-Seq Library Preparation Kits* use a unique terminal-tagging technology* that simplifies the preparation of directional, paired-end libraries for Illumina® sequencing. The kits are suitable for any animal, plant, or bacterial species.
• Start with 50 ng or less of rRNA-depleted or poly(A)-enriched RNA.• Cluster-ready, directional libraries in about 3 hours without adaptor ligation.• Use Illumina-compatible barcodes (available separately) or user-defined barcodes.• Libraries demonstrate high concordance with MAQC qPCR data.• High percentage of mapped reads from intact, partially degraded, and FFPE RNA
samples.*Covered by issued and/or pending patents.
A. Intact UHRR and BrRR
Correlation between data obtained from ScriptSeq™ libraries and corresponding MAQC qPCR data. A) Libraries prepared from rRNA-depleted, intact UHRR and BrRR. B) Libraries prepared from partially fragmented, rRNA-depleted UHRR and BrRR. UHRR, Universal Human Reference RNA; BrRR, Brain Reference RNA.
Figure1.Correlationofgeneexpression.
B. Fragmented UHRR and BrRR
18 www.epicentre.com
Cloning
CopyControl™ Fosmid Library Production KitSave time and effort constructing a fosmid library
The CopyControl Fosmid Library Production Kits* clone randomly sheared DNA to produce unbiased genomic libraries containing 35- to 40-kb inserts. The unique CopyControl technology enables you to grow clones at single-copy number to ensure insert stability and cloning of expressed toxic sequences, and then induce the clones to high-copy number (20-50 copies per cell) for high yields of DNA.
• Kits include all the components required to construct 10 complete libraries. • Random shearing of genomic DNA ensures more complete and unbiased libraries
compared to partial restriction digestion.*Covered by issued and/or pending patents.
Cat.# Quantity
CopyControl™ Fosmid Library Production KitCCFOS110 1 KitComplete kit for constructing a CopyControl Fosmid library in the Cloning-Ready pCC1FOS Vector.
CopyControl™ HTP Fosmid Library Production KitCCFOS059 1 KitComplete kit for constructing a CopyControl Fosmid library in the Cloning-Ready pCC2FOS Vector.
Once the library has been prepared, individual clones can be cultured in small volumes and induced to multiple-copy number for high yields of high-purity DNA using Epicentre’s DirectLysis Fosmid96 Kit or FosmidMAX™ DNA Purification Kit.
Figure1.OverviewoftheprocessfortheCopyControl™Kits.
[email protected] • (800) 284-8474 19
Other Reagents
Ready-Lyse™ Lysozyme Solution Purify nucleic acids or protein from bacteria
Ready-Lyse™ Lysozyme Solution is a recombinant lysozyme preparation, derived from a nonmammalian, nonavian source, that is useful for the lysis of Gram-negative and Gram-positive bacteria. The specific activity of Ready-Lyse Lysozyme is 200-fold higher than that of egg-white lysozyme and, therefore, less lysozyme is needed in a reaction. Unlike egg-white lysozyme, Ready-Lyse Lysozyme Solution is stable at –20°C, eliminating the need to prepare a fresh solution for each use.
Unit Definition: One unit of Ready-Lyse Lysozyme produces a decrease in A350 of 0.001 per minute at 23°C with a 0.5 mg/ml suspension of lyophilized E. coli K802 cells in 50 mM Tris-HCl (pH 7.5).
Cat.# Quantity
Ready-Lyse™ Lysozyme SolutionR1802M 2 X 106 Units R1804M 4 X 106 UnitsR1810M 10 X 106 Units
Proteinase K Remove protein from nucleic acid preps
Proteinase K is used to digest protein and remove contamination from preparations of nucleic acid. The addition of Proteinase K to nucleic acid preparations rapidly inactivates nucleases that might otherwise degrade the DNA or RNA during purification. Proteinase K remains active under denaturing conditions, such as the presence of SDS or urea, chelating agents (EDTA, sulfhydryl reagents), and trypsin or chymotrypsin inhibitors.
Cat.# Concentration Quantity
Proteinase KMPRK092 50 µg/µl 2 ml